1. Field of the Invention
This invention relates generally to sodium channel proteins and more particularly to a novel cloned xcex1-subunit of a voltage-gated, tetrodotoxin-sensitive sodium channel protein. This invention further relates to the production of this protein by recombinant technology and nucleic acid sequences encoding for this protein.
2. Previous Art
The basic unit of information transmitted from one part of the nervous system to another is a single action potential or nerve impulse. The xe2x80x9ctransmission linexe2x80x9d for these impulses is the axon, or nerve fiber. The electrical excitability of the nerve membrane has been shown to depend on the membrane""s voltage-sensitive ionic permeability system that allows it to use energy stored in ionic concentration gradients. Electrical activity of the nerve is triggered by a depolarization of the membrane, which opens channels through the membrane that are highly selective for sodium ions, which are then driven inward by the electrochemical gradient. Of the many ionic channels, the voltage-gated or voltage-sensitive sodium channel is one of the most studied. It is a transmembrane protein that is essential for the generation of action potentials in excitable cells. An excellent review of sodium channels is presented in Catterall, TINS 16(12):500-506 (1993).
The cDNAs for several Na+ channels have been cloned and sequenced. Numa, et al., Annals of the New York Academy of Sciences 479:338-355 (1986), describe cDNA from the electric organ of eel and two different cDNAs from rat-brain. Rogart, U.S. Pat. No. 5,380,836, describes cDNA from rat cardiac tissue. See also Rogart, Cribbs, et al., Proc. Natl. Acad. Sci., 86:8170-8174 (1989). A peripheral nerve sodium channel, referred to as PN1, has been detected based on sodium current studies and hybridization to a highly conserved sodium channel probe by D""Arcangelo, et al., J. Cell Biol. 122:915-921 (1993), and subsequently cloned from PC12 cells, Toledo-Aral, et al., Proc. Nat. Acad. Sci. 94:1527-1532 (1997). The sequence of rat PN1 cloned from dorsal root ganglia and its functional expression have been described, Sangameswaran, et al., J. Biol. Chem 272:14805-14809 (1997). Other cloned sodium channels include rat brain types IIa, Auld, et al., Neuron 1:449-461 (1988), and III, Kayano, et al., FEBS Lett. 228:187-194 (1988), rat skeletal muscle, Trimmer, et al., Neuron 3:33-49 (1989), rat NaCh6, Schaller, et al., J. Neurosci. 15:3231-3242 (1995), rat peripheral nerve sodium channel type 3 (rPN3), Sangameswaran, et al., J. Biol Chem. 271:5953-5956 (1996), also called SNS, Akopian, et al., Nature 379:257-262 (1996), rat atypical channel, Felipe, et al., J. Biol. Chem. 269:30125-30131 (1994), and the rat glial sodium channel, Akopian, et al., FEBS Lett. 400:183-187 (1997).
These studies have shown that the amino acid sequence of the Na+ channel has been conserved over a long evolutionary period. These studies have also revealed that the channel is a single polypeptide containing four internal repeats, or homologous domains (domains I-IV), having similar amino acid sequences. Each domain folds into six predicted transmembrane xcex1-helices or segments: five are hydrophobic segments and one segment is highly charged with many lysine and arginine residues. This highly charged segment is the fourth transmembrane segment in each domain (the S4 segment) and is likely to be involved in voltage-gating. The positively charged side chains on the S4 segment are likely to be paired with the negatively charged side chains on the other five segments, such that membrane depolarization could shift the position of one helix relative to the other, thereby opening the channel. Accessory subunits may modify the function of the channel.
Therapeutic utilities in recombinant materials derived from the DNA of the numerous sodium channels have been discovered. For example, Cherksey, U.S. Pat. No. 5,132,296, discloses purified Na+ channels that have proven useful as therapeutic and diagnostic tools.
Isoforms of sodium channels are divided into xe2x80x9csubfamiliesxe2x80x9d. The term xe2x80x9cisoformxe2x80x9d is used to mean distinct but closely related sodium channel proteins, i.e., those having an amino acid homology of approximately 60-80%. These isoforms also show strong homology in functions. The term xe2x80x9csubfamiliesxe2x80x9d is used to mean distinct sodium channels that have an amino acid homology of approximately 80-95%. Combinations of several factors are used to determine the distinctions within a subfamily, for example, the speed of a channel, chromosomal location, expression data, homology to other channels within a species, and homology to a channel of the same subfamily across species. Another consideration is an affinity to tetrodotoxin (xe2x80x9cTTXxe2x80x9d). TTX is a highly potent toxin from the puffer or fugu fish which blocks the conduction of nerve impulses along axons and in excitable membranes of nerve fibers. TTX binds to the Na+ channel and blocks the flow of sodium ions.
Studies using TTX as a probe have shed much light on the mechanism and structure of Na+ channels. There are three Na+ channel subtypes that are defined by the affinity for TTX, which can be measured by the IC50 values: TTX-sensitive Na+ channels (IC50≈1-30 nM), TTX-insensitive N+ channels (IC50≈1-5 xcexcm), and TTX-resistant Na+ channels (IC50xe2x89xa7100 xcexcM).
TTX-insensitive action potentials were first studied in rat skeletal muscle. See Redfern, et al., Acta Physiol. Scand. 82:70-78 (1971). Subsequently, these action potentials were described in other mammalian tissues, including newborn mammalian skeletal muscle, mammalian cardiac muscle, mouse dorsal root ganglion cells in vitro and in culture, cultured mammalian skeletal muscle, and L6 cells. See Rogart, Ann. Rev. Physiol. 43:711-725 (1981).
Dorsal root ganglia neurons possess both TTX-sensitive (IC50 ≈0.3 nM) and TTX-resistant (IC50≈100 xcexcM) sodium channel currents, as described in Roy, et al., J. Neurosci. 12:2104-2111 (1992).
TTX-resistant sodium currents have also been measured in rat nodose and petrosal ganglia, Ikeda, et al., J. Neurophysiol. 55:527-539 (1986) and Stea, et al., Neurosci. 47:727-736 (1992).
One aspect of the present invention is a purified and isolated DNA sequence encoding for a novel TTX-sensitive sodium channel protein, in particular, the xcex1-subunit of this protein. Another embodiment of the invention is a purified and isolated DNA sequence encoding for a splice variant of the novel TTX-sensitive sodium channel.
Another aspect of the invention is a method of stabilizing the full length cDNA which encodes the protein sequence of the invention.
Also included in this invention are alternate DNA forms, such as genomic DNA, DNA prepared by partial or total chemical synthesis from nucleotides, and DNA having deletions or mutations.
Another aspect of the invention is a novel probe based on known sodium channels for screening rat cDNA libraries.
Further aspects of the invention include expression vectors comprising the DNA of the invention, host cells transformed or transfected by these vectors, and clonal cell lines expressing the DNA of the invention. Also disclosed is the cDNA and MRNA derived from the DNA sequences of the invention.
Another aspect of the present invention are recombinant polynucleotides and oligonucleotides comprising a nucleic acid sequence derived from the DNA sequence of this invention.
Still another aspect of the invention is the novel rat TTX-sensitive sodium channel protein and fragments thereof, encoded by the DNA of this invention.
Further provided is a method of inhibiting the activity of the novel TTX-sensitive sodium channel comprising administering an effective amount of a compound having an IC50 of 1 nM or less.
Also forming part of this invention is an assay for inhibitors of the sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel.
Another part of this invention is a method of employing the DNA for forming monoclonal and polyclonal antibodies, for use as molecular targets for drug discovery, highly specific markers for specific antigens, detector molecules, diagnostic assays, and therapeutic uses.